Many genes produce multiple proteins by alternative mRNA processing. The long-term goal of the project is to understand the molecular basis for the regulated splicing of the rat fibronectin gene. Fibronectins are a family of large glycoproteins found in extracellular matrices and blood. The single fibronectin (FN) gene encodes multiple mRNAs from a single pre-mRNA via alternative splicing at three positions. FN alternative splicing is developmentally regulated in a cell-specific manner, and changes in splicing occur during the ECM remodeling that accompanies wound healing, fibrosis, and tumorigenesis. The large FN gene presents other challenges to the splicing apparatus and serves as an attractive model for studies of RNA processing. The primary goal of the proposed experiments is to understand the molecular basis of regulated alternative-splicing of the EIIIB exon of the rat FN gene. It was predicted that first, the pre-mRNA should contain sequences that regulate splicing, and second, cell-specific factors should recognize the cis-elements. Initial studies are consistent with these predictions. The intron upstream of alternative exon EIIIB, IVS1, is poorly recognized by the splicing machinery in vitro, apparently due to sequences that lie upstream of EIIIB. Several-nuclear factors bind specifically within this region. Completion of Specific Aims 1-3 should lead to a detailed description of the cis and trans requirements for EIIIB splicing. Specific Aim 1 is to identify the sequences responsible for the inefficient, conditional in vitro processing of IVS1 by mutagenesis and subsequent functional studies. The effect mutations in IVS1 on EIIIB selection will be evaluated in cells that normally include or exclude the exon. In vitro analyses of the same mutants will be performed using nuclear extract prepared from the different cells. UV crosslinking experiments have identified several IVS1-binding proteins, including U2AF and PTB. Specific Aim 2 is to continue these studies, and to use purified proteins and specifically depleted extracts to clarify the functions of putative regulators of splicing. Furthermore, analyses of extracts prepared from non-HeLa cells will correlate the level of EIIIB inclusion with the relative amounts of various splicing factors. Specific Aim 3 is to investigate the role of competition between the 3' splice site of exon III8a in IVS2 and the EIIIB 3' splice site in IVS1 on EIIIB selection. Additionally, mutagenesis of sequences within EIIIB will localize sequences that negatively affect exon selection; proteins that bind such an exonic element will be sought. Finally, Specific Aim 4 is the identification of features of the rat FN gene involved in the selection of non-canonical 3' splice sites and the large V region exon. Two constitutive FN exons have 3' splice sites that deviate from the consensus; it is predicted that these sites will recognized inefficiently by the splicing apparatus. Sequences that compensate for such naturally occurring weak sites will be identified. The entire alternatively spliced V exon is unusually large. As current models of exon recognition do not accommodate large exons, features that permit recognition of V will be characterized.